Single potential electrophoresis microchip with reduced bias using pressure pulse injection

We propose two variants of a new injection technique for use in electrophoresis microchips, called "front gate pressure injection" and "back gate pressure injection", that both enable a controlled and reproducible sample introduction with reduced bias compared to electrokinetic gated injection. A continuous flow of a test solution of fluorescein/rhodamine B in 20 mM Tris/boric acid buffer (pH 8.6) sample test solution is electrokinetically driven near to the entrance of the separation channel, using a single voltage (3 kV) that is constant in time. A sample plug is injected in the separation channel by a pressure pulse of the order of 0.1 s. The latter is generated using the mechanical deflection of a PDMS membrane that is loosely placed on a dedicated chip reservoir. The analysis of the peak area ratio of the separated compounds demonstrates a nearly constant sample composition when using pressure-based injection. A small remaining injection bias for the shortest membrane deflection times can be attributed to a dilution effect of the charged compound due to the presence of an electrical field transverse to the sample flow boundary in the channel junction.     

Characterization of various geometric arrangements of “air-assisted” flow gating interfaces for capillary electrophoresis

For connecting flow-through analytical methods with capillary electrophoresis, a chip working in the air-assisted flow gating interface regime is cast from poly(dimethylsiloxane). In the injection space, the exit from the delivery capillary is placed close to the entrance to the separation capillary. Prior to injecting the sample into the separation capillary, the background electrolyte is forced out of the injection space by a stream of air. In the empty space, a drop of the sample with a volume of <100 nL is formed between the exit from the delivery capillary and the entrance into the separation capillary, from which the sample is injected hydrodynamically into the separation capillary. After injection, the injection space is filled with BGE, and the separation can be begun. Three geometric variants for the mutual geometric arrangement of the delivery and separation capillaries were tested: the delivery capillary is placed perpendicular to the separation capillary, from either above or below, or the capillaries are placed axially, that is, directly opposite one another. All of the variants are equivalent from the analytical and separation efficiency viewpoints. The repeatability expressed by RSD is up to 5%. The tested flow gating interface variants are also suitable for continuous and discontinuous sampling at flow rates of the order of units of μL/min. The developed instrument for sequential electrophoretic analysis operates fully automatically and is suitable for rapid sequential monitoring of dynamic processes.     

食品中主要霉菌毒素分析方法的研究进展

食品中霉菌毒素的检测面临基质复杂、污染浓度水平低的困扰,研发选择性高、富集能力强的样品前处理方法和高灵敏的分析方法对于提升食品中霉菌毒素的检测能力、保障食品安全具有重要意义。该文综述了近年来食品中霉菌毒素分析方法的研究进展,并对其发展方向作总结和展望。     

微流控芯片技术在生命科学研究中的应用

微流控芯片最初起源于分析化学领域,是一种采用精细加工技术,在数平方厘米的基片,制作出微通道网络结构及其它功能单元,以实现集微量样品制备、进样、反应、分离及检测于一体的快速、高效、低耗的微型分析实验装置.随着微电子及微机械制作技术的不断进步,近年来微流控芯片技术发展迅猛,并开始在化学、生命科学及医学器件等领域发挥重要作用.本文首先简单介绍了微流控芯片制作材料和工艺,然后主要阐述了其在蛋白质分离、免疫分析、DNA分析和测序、细胞培养及检测等方面的应用进展.     

Fully integrated PDMS/SU-8/quartz microfluidic chip with a novel macroporous poly dimethylsiloxane (PDMS) membrane for isoelectric focusing of proteins using whole-channel imaging detection

A fully integrated polydimethylsiloxane (PDMS)/modified PDMS membrane/SU-8/quartz hybrid chip was developed for protein separation using isoelectric focusing (IEF) mechanism coupled with whole-channel imaging detection (WCID) method. This microfluidic chip integrates three components into one single chip: (i) modified PDMS membranes for separating electrolytes in the reservoirs from the sample in the microchannel and thus reducing pressure disturbance, (ii) SU-8 optical slit to block UV light (below 300?nm) outside the channel aiming to increase detection sensitivity, and (iii) injection and discharge capillaries for continuous operation. Integration of all these components on a single chip is challenging because it requires fabrication techniques for perfect bonding between different materials and is prone to leakage and blockage. This study has addressed all the challenges and presented a fully integrated chip, which is more robust with higher sensitivity than the previously developed IEF chips. This chip was tested by performing protein and pI marker separation. The separation results obtained in this chip were compared with that obtained in commercial cartridges. Side-by-side comparison validated the developed chip and fabrication techniques.     

芯片毛细管电泳用于人乳头瘤病毒分析的研究进展

人乳头瘤病毒（human papillomavirus,HPV）是一种常见的球形DNA病毒,目前已报道其可以导致6种类型的癌症发生,因此HPV病毒检测方法的研究引起了人们的重视。芯片毛细管电泳（MCE）,作为一种芯片实验设备,结合各种信号放大技术为HPV分型检测提供了简单、快速、高灵敏度和易便携化的检测方法。该文综述了MCE在常规HPV分型检测中的最新研究进展,主要分为MCE技术和MCE结合核酸扩增技术两个部分。综述的第一部分介绍了MCE系统、MCE芯片结构设计和电泳分离方法。典型的MCE系统包含了高压电源、分离芯片、电解液池、进样系统、检测系统等。该文还介绍了近年来应用最广泛的4种芯片通道,包括分离直通道、T型通道、蛇形通道以及双通道,并分别对它们的优缺点进行了比较。第二部分主要介绍芯片电泳在HPV检测中的应用和发展。由于MCE技术的应用,HPV目标物的分离时间,从以前的几个小时缩短到几分钟,极大地提高了分离速度。重点介绍了各种核酸扩增技术结合MCE检测HPV的方法。对聚合酶链式反应（PCR）和MCE结合用于HPV的检测技术、环介导等温扩增（LAMP）技术的HPV检测方法、基于PCR结合限制性片段长度多态性（RFLP）技术用于HPV分型的DNA检测、基于核酸序列扩增（NASBA）技术检测HPV mRNA、巢式PCR等进行了比较分析。其次,对HPV其他检测方法进行了总结,其中包括PCR结合傅里叶变换红外光谱法（FT-IR）、纳米技术、DNA探针结合电化学方法、亚铜粒子氧化还原锌掺杂的二硫化钼量子点结合T7外切酶电化学发光法和基于CRISPR/Cas12a的环介导等温扩增法。在这些非MCE方法中,电化学传感法,如阻抗法、脉冲伏安法和流动生物传感器,由于背景信号低、时间控制能力强,是一种比较理想的方法。最后,虽然近年来MCE技术得到了发展,所开发的设备得到了应用,但目前在MCE技术、方法和应用方面仍然存在一些挑战。MCE技术在HPV分型检测应用中面临的第一个挑战是,MCE本身无法对HPV核酸进行信号放大,从而不能在HPV的高灵敏和高选择性分析中得到很好的应用。第二个挑战是,虽然有一些研究者已经成功地将PCR和MCE集成在一个芯片上,但该技术的广泛应用仍面临困难,目前仍然没有真正集成的PCR-MCE芯片用于HPV检测。第三个挑战是目前MCE技术无法实现小型化、自动化器件的制造。最后,文章就MCE在HPV分型检测中开发更自动化、更快速以及更稳定可靠的检测技术提出了一些观点和见解,希望能对感兴趣的读者提供一些启发。     

High-resolution DNA separation in microcapillary electrophoresis chips utilizing double-L injection techniques

An experimental and numerical investigation into the use of high-resolution injection techniques to separate DNA fragments within electrophoresis microchips is presented. The principal material transport mechanisms of electrokinetic migration, fluid flow, and diffusion are considered, and several variable-volume injection methods are discussed. A detailed analysis is provided of a double-L injection technique, which employs appropriate electrokinetic manipulations to reduce sample leakage within the microchip. The leakage effect in electroosmotic flow (EOF) is investigated using a sample composed of rhodamine B and Cy3 dye. Meanwhile, the effects of sample leakage in capillary electrophoresis (CE) separation are studied by considering the separation of 100-base pairs (bp) DNA ladders and HaeIII-digested PhiX-174 DNA samples. The present experimental and simulation results indicate that the unique injection system employed in the current microfluidic chip has the ability to replicate the functions of both the conventional cross-channel and the shift-channel injection systems. Furthermore, applying the double-L injection method to these two injection systems is shown to reduce sample leakage significantly. The proposed microfluidic chip and double-L injection technique developed in this study have an exciting potential for use in high-resolution, high-throughput biochemical analysis applications and in many other applications throughout the micrototal analysis systems field.     

In-column field-amplified sample stacking of biogenic amines on microfabricated electrophoresis devices

A novel method for performing in-column field-amplified sample stacking (FASS) in chip-based electrophoretic systems is presented. The methodology involves the use of a narrow sample channel (NSC) injector. NSC injectors allow sample plugs to be introduced directly into the separation channel, and subsequent stacking and separation can proceed without any need for leakage control. More importantly, stacking and separation occur in a single step negating the requirement for complex channel geometries and voltage switching to control sample plugs during the stacking procedure. The chip is composed of six paralleled systems. Using the NSC injector design, the number of reservoirs in the multiplexed chip is reduced to N + 2, where N is the number of paralleled systems. This design feature radically reduces the complexity in chip structures and associated chip operation. The approach is applied to the analysis of fluorescently labelled biogenic amines affording detection at concentrations down to 20 pM.     

Determination of bromate in drinking water by zone electrophoresis-isotachophoresis on a column-coupling chip with conductivity detection

The use of capillary zone electrophoresis (CZE) on-line coupled with isotachophoresis (ITP) sample pretreatment (ITP-CZE) on a poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors, to the determination of bromate in drinking water was investigated. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the ITP-CZE separations. A high sample load capacity, linked with the use of ITP in this combination, made possible loading of the samples by a 9.2 microL sample injection channel of the chip. In addition, bromate was concentrated by a factor of 10(3) or more in the ITP stage of the separation and, therefore, its transfer to the CZE stage characterized negligible injection dispersion. This, along with a favorable electric conductivity of the carrier electrolyte solution, contributed to a 20 nmol/L (2.5 ppb) limit of detection for bromate in the CZE stage. Sample cleanup, integrated into the ITP stage, effectively complemented such a detection sensitivity and bromate could be quantified in drinking water matrices when its concentration was 80 nmol/L (10 ppb) or slightly less while the concentrations of anionic macroconstituent (chloride, sulfate, nitrate) in the loaded sample corresponding to a 2 mmol/L (70 ppm) concentration of chloride were still tolerable. The samples containing macroconstituents at higher concentrations required appropriate dilutions and, consequently, bromate in these samples could be directly determined only at proportionally higher concentrations.     

Parallel analysis of biomolecules on a microfabricated capillary array chip

This paper focused on a self-developed microfluidic array system with microfabricated capillary array electrophoresis (mu-CAE) chip for parallel chip electrophoresis of biomolecules. The microfluidic array layout consists of two common reservoirs coupled to four separation channels connected to sample injection channel on the soda-lime glass substrate. The excitation scheme for distributing a 20 mW laser beam to separation channels in an array is achieved. Under the control of program, the sample injection and separation in multichannel can be achieved through six high-voltage modules' output. A CCD camera was used to monitor electrophoretic separations simultaneously in four channels with LIF detection, and the electropherograms can be plotted directly without reconstruction by additional software. Parallel multichannel electrophoresis of series biomolecules including amino acids, proteins, and nucleic acids was performed on this system and the results showed fine reproducibility.     

Sample pre-concentration by isotachophoresis in microfluidic devices

We have designed microfluidic devices with the aim of coupling isotachophoresis (ITP) with zone electrophoresis (ZE) as a method to increase the concentration limit of detection in microfluidic devices. We used plastic multi-channel chips, designed with long sample injection channel segments, to increase the sample loading. The chip was designed to allow stacking of the sample into a narrow band by discontinuous ITP buffers and subsequent separation in the ZE mode. In the ITP-ZE mode, with a 2-cm long sample injection plug, sensitivity was increased by 400-fold over chip ZE and we found that the separation performance after the ITP stacking was comparable to that of regular chip ZE. We report sub-picomolar limits of detection of fluorescently labeled ACLARA eTag reporter molecules electrokinetically injected from cell lysate sample matrixes containing moderate salt concentrations. We evaluated sample injections from buffers with varied ionic strengths and found that efficient stacking and separations were obtained in both low and high conductivity buffers, including physiological buffer with at least 140 mM salt. We applied ITP-ZE to the analysis of a cell surface protease (ADAM 17) which used live intact cells in physiological buffers with detection limits below 10 cells/assay.     

Capillary electrophoresis of single mammalian cells

Capillary electrophoresis (CE) has been established as powerful tool for single cell analysis. Newly developed sampling, separation and detection methods have allowed the investigation of single mammalian cells with CE despite their small size and complex composition. Advances in sample injection techniques include several novel methods for the injection of whole cells and sampling techniques for the study of cellular secretion. CE of single mammalian cells has been applied in a wide range of fields including protein analysis, neuroscience, and oncology. The development of new detection schemes in the analysis of single mammalian cells with CE has included studies of protein expression and the utilization of mass spectrometric and electrochemical detection. Subcellular mammalian cell analysis with CE also has been investigated.     

微流控芯片测定单细胞内化学组分的进展

细胞是生命的基本单元。由于细胞的个体差异,传统分析群体细胞的方法难以得到单细胞的重要信息。准确可靠地测定单细胞内化学组分的含量能大大提高从正常细胞中辨别不正常细胞的能力,为进一步研究和发展生物化学、医学和临床检验等领域奠定基础。近年来,用微流控芯片进行单细胞分析已引起广泛的兴趣。微流控芯片可以集成单细胞进样、溶膜、电泳分离胞内化学组分和高灵敏度测定等一系列操作步骤,为分析单细胞内的化学组分提供了新的技术平台。本文主要综述了近年来微流控芯片测定单细胞内化学组分的进展。重点在于利用电渗流、压力结合电渗流和激光镊子等技术操控单细胞在微流控芯片上完成单细胞进样、溶膜、细胞内化学组分的电泳分离和高灵敏度测定等一系列操作步骤。对在微流控芯片上的衍生技术也做了较为详细的阐述。     

芯片液相色谱技术进展

微型化是现代分析仪器发展的重要趋势.微型化液相色谱仪器在提供与常规尺度液相色谱相同甚至更高分离效率的同时,可以有效减少溶剂和样品的消耗;在液相色谱-质谱联用中,低流速进样可以有效提高质谱离子源的离子化效率,提高质谱检测效率;对于极微量样品的分离,微型化的液相色谱可以有效减少样品稀释;液相色谱的微型化还有利于液相色谱仪器...     

Purification and partial characterization by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the recombinant transposase,TniA

This work deals with zone electrophoresis (ZE) separations of proteins on a poly(methyl methacrylate) chip with integrated conductivity detection. Experiments were performed in the cationic mode of the separation (pH 2.9) with a hydrodynamically closed separation compartment and suppressed electroosmotic flow. The test proteins reached the detector in less than 10 min under these working conditions and their migration times characterized excellent repeatabilities (0.1–0.6% RSD values). The chip-to-chip agreements of the migration times, evaluated from the ZE runs performed on three chips, were within 1.5%. The conductivity detection provided for protein, loaded on the chip at 10–1000 μg/ml concentrations, detection responses were characterized by 1–5% RSD values of their peak areas. Such migration and detection performances made a frame for reproducible baseline separations of a five-constituent mixture (cytochrome c, avidin, conalbumin, human hemoglobin and trypsin inhibitor). On the other hand, a high sample injection channel/separation compartment volume ratio of the chip (500 nl/8500 nl) restricted the resolution of proteins of very close effective mobilities in spite of the fact that in the initial phase of the separation an electric field stacking was applied. A maximum macroconstituent/trace constituent ratio attainable for proteins on the chip was assessed for cytochrome c (quantifiable when its concentration in the loaded sample was 10 μg/ml) and apo-transferrin (containing a trace constituent migrating in the position of cytochrome c detectable when the load of apo-transferrin was 2000 μg/ml). This assessment indicated that a ratio of 1000:1 is attainable with the aid of conductivity detection on the present chip.     

On-line immunoaffinity capillary electrophoresis based on magnetic beads for the determination of alpha-1 acid glycoprotein isoforms profile to facilitate its use as biomarker

An immunoaffinity purification method coupled on-line to capillary electrophoresis (IACE) which allows the determination of several isoforms of intact alpha-1 acid glycoprotein (AGP) in serum samples using UV detection is developed. The immunoaffinity step is based on anti-AGP antibodies (Abs) covalently bound to magnetic beads (MBs) which are captured at the inlet end of the capillary using permanent magnets placed inside the cartridge of the CE instrument. The on-line method includes injection of the MBs with the Ab bound (MBs–Ab) and their trapping by the magnets at the entrance of the separation column, injection of serum sample and capture of AGP by the Abs, release of captured AGP, focus of desorbed protein, separation of AGP isoforms, and removal of MBs–Ab. The optimization of the different factors involved in each step allowed purification, separation and detection of AGP isoforms in a single electrophoretic analysis in about 1 h. Automation, sample and reagents consumption as well as analysis time was improved compared to off-line alternatives which use purification of AGP in an immunochromatographic column and CE separation of AGP isoforms in two independent operations. The analytical methodology developed allows the separation of 10 AGP isoforms in serum samples from a healthy donor. For a serum sample, precision (expressed as relative standard deviation) in terms of corrected area percentage was better than 0.5% for each peak accounting for more than 10% of total AGP and it was better than 4.0% in terms of relative migration time of each AGP isoform considering the whole process.     

Determination of oxalate in beer by zone electrophoresis on a chip with conductivity detection

The use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel combined with a 500 nL sample injection channel) and a pair of on‐chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in beer was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (3.8), implemented by aspartic acid and bis‐tris propane, provided an adequate selectivity in the separation of oxalate from anionic beer constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 0.5 μmol/L concentration also in samples containing chloride (a major anionic constituent of beer) at a 1800 higher concentration. Such a favorable analyte/matrix concentration ratio made possible accurate and reproducible [typically, 2–5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample] determination of oxalate in 500 nL volumes of 20–50‐fold diluted beer samples. Short analysis times (about 200 s), minimum sample preparation, and reproducible migration times of this analyte (0.5–1.0% RSD values) were characteristic for ZE on the chip.     

Multiple‐injection high‐throughput gas chromatography analysis

Multiple‐injection techniques have been shown to be a simple way to perform high‐throughput analysis where the entire experiment resides in a single chromatogram, simplifying the data analysis and interpretation. In this study, multiple‐injection techniques are applied to gas chromatography with flame ionization detection and mass detection to significantly increase sample throughput. The unique issues of implementing a traditional “Fast” injection mode of multiple‐injection techniques with gas chromatography and mass spectrometry are discussed. Stacked injections are also discussed as means to increase the throughput of longer methods where mass detection is unable to distinguish between analytes of the same mass and longer retentions are required to resolve components of interest. Multiple‐injection techniques are shown to increase instrument throughput by up to 70% and to simplify data analysis, allowing hits in multiple parallel experiments to be identified easily.     

Determination of oxalate in urine by zone electrophoresis on a chip with conductivity detection

The use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel coupled to a 500 nL sample injection channel) and a pair of on-chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in urine was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (4.0) provided an adequate selectivity in the separation of oxalate from anionic urine constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 8 x 10(-8) mol/L concentration also in samples containing chloride (a major anionic constituent of urine) at 3.5 x 10(-3) mol/L concentrations. Such a favorable analyte/matrix concentration ratio (in part, attributable to a transient isotachophoresis stacking in the initial phase of the separation) made possible accurate and reproducible (typically, 2-5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample) determination of oxalate in 500 nL volumes of 20-100-fold diluted urine samples. Short analysis times (about 280 s), no sample pretreatment (not considering urine dilution) and reproducible migration times of this analyte (0.5-1.0% RSD values) were characteristic for ZE on the chip. This work indicates general potentialities of the present chip design in rapid ZE analysis of samples containing the analyte(s) at high ionic matrix/analyte concentration ratios.